title: High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays (CAPPIA).
abstract: We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of microarrays. Nanoliters of samples containing mixtures of bait and prey expression plasmids together with an autofluorescent reporter are immobilized on glass slides in defined array formats and air-dried. Subsequently, monolayers of adherent mammalian cells are grown on these slides so that only cell clusters on top of each feature become transfected, whereas the surrounding cells remain untransfected. If the expressed proteins show any interaction, the bait and prey proteins inside the cells are functionally linked together at the promoter of the autofluorescent reporter, reconstituting transcriptional activity, and cells become fluorescent. The cluster of cells that express that particular combination of bait and prey constructs can be identified by its position in the array by simple fluorescence detection using common DNA array scanners or high-throughput microscopy. CAPPIA allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. The high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.
authors.affiliation: andrea.fiebitz@charite.de
authors.forename: Andrea
authors.initials: A
authors.lastname: Fiebitz
authors.forename: Dominique
authors.initials: D
authors.lastname: Vanhecke
mesh_terms: D059002:Androgen Receptor Antagonists / Q000494:pharmacology; D000728:Androgens / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D006801:Humans; D008024:Ligands; D015741:Metribolone / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011327:Printing; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046888:Tissue Array Analysis / Q000379:methods*; D014162:Transfection; D020798:Two-Hybrid System Techniques / Q000295:instrumentation*
chemical_list: D059002:Androgen Receptor Antagonists; D000728:Androgens; D008024:Ligands; D011944:Receptors, Androgen; D015741:Metribolone